Bi-and trispecific antibodies for the induction of anti-tumor immunity

ABSTRACT

According to the invention, a method is provided wherein intact bispecific or trispecific antibodies which at the same time bind to the T cell receptor complex of a T cell, to tumour-associated antigens on a tumour cell, and, via the Fc portion of the bispecific antibody, to Fc receptor-positive cells are used for the induction of an anti-tumour immunity in humans and animals.

[0001] The invention relates to the use of intact bispecific antibodiesfor the induction of anti-tumour immunity in humans and animals.

[0002] Despite of the progress in chemotherapy and radiotherapy achievedin recent years, malignant diseases in humans, for example terminalbreast cancer, still have an extraordinarily unfavourable prognosis.These diseases are impossible to cure. Therefore, it is necessary todevelop new treatment strategies. In this respect, great hopes areplaced in immunotherapeutical approaches enabling the immune system ofthe patient to reject the tumour. It is well-known thattumour-associated antigens exist on tumour cells and that in principlethe immune system can recognize these antigens and attack the malignantcells. Tumours have, however, developed certain strategies enabling themto escape the immune reaction for example by insufficient presentationof tumour-associated antigens and/or insufficient activation of thetumour-specific T cells which are generally present.

[0003] With about 43.000 new cases/year, breast cancer occupies a topposition in the cancer statistics of women in Germany. Less than onethird of the women suffering from lymph node invasion at the time ofdiagnosis survive for 10 years without relapse. Against this background,attempts have been made since several years towards the prolongation oflife or even healing of female patients suffering from extensive lymphnode invasion and distant metastases by means of autologous bone marrowand stem cell transplantation in connection with high-dose chemotherapy.Despite high response rates to the high-dose chemotherapy a permanentcure in the metastatic stage rarely occurs.

[0004] To date, immunotherapeutic approaches to the treatment of mammacarcinoma have been restricted to methods for unspecific stimulationsuch as the treatment with BCG or levamisole as well as the use of LAKcells and NK cells together with IL-2 (15, 16). There has been noevidence for a prolongation of life by the types of immunotherapy usedso far, while the treatment with BCG rather turned out to bedisadvantageous (15). Since the unspecific activation of cells hasachieved little success also with other tumour types, attempts were madeto raise a specific immune reaction.

[0005] For example, T cell-redirecting bispecific antibodies have beenused in tumour therapy. These antibodies are able to bind to a T cellreceptor complex by one binding arm and to a tumour-specific antigen ona tumour cell by the second binding arm. Due to resulting activation ofthe T cell and the spatial proximity of the tumour cell, the latter iskilled by either by induction of apoptosis or by cytokins such as TNF-αor perforin.

[0006] It is an object of the present invention to provide a novelmethod for the therapy of malignant diseases in humans.

[0007] According to the invention, this object has been achieved by thefeatures characterized in more detail in claim 1. Preferred embodimentsof the invention are obvious from the dependent Claims.

[0008] Thus, the invention discloses a method for the induction of ananti-tumor immunity by administering to a human or an animal subject anefficient amount of an intact bispecific or trispecific antibody or acombination thereof, having the following properties and effects of:

[0009] (a) binding to a T cell to which it mediates a first activationsignal;

[0010] (b) binding to tumour-associated antigens on a tumour cell;

[0011] (c) binding, by its Fc portion (in the case of bispecificantibodies) or a third specificity (in the case of trispecificantibodies), to the Fc receptor of Fc receptor-positive cells;

[0012] (d) activation of the Fc receptor-positive cell by binding to theFc receptor-positive cell and, thereby, initiating or increasing theexpression of cytokins and/or costimulatory antigens;

[0013] (e) transfer of at least a second activation signal required forphysiological activation of the T cell to the T cell by theco-stimulatory antigens and/or cytokins, this activation being indicatedby up-regulation of activation markers, killing of the tumour cell,and/or T cell proliferation.

[0014] Preferably, the antibodies used in the method of the presentinvention are able to activate the tumour-specific T cells recognizing atumour-specific peptide presented on the tumour cells by MHC class Iand/or class II via their T cell receptor upon binding to the bispecificor trispecific antibody as described under (e).

[0015] Further, the antibodies used according to the invention are ableto reactivate the tumour-specific T cells being in an anergic state.Furthermore, they are able to induce tumour-reactive complement-bindingantibodies and, thus, induce a humoral immune reaction.

[0016] Binding to the T cell takes place via CD3, CD2, CD5, CD28, and/orCD44. The Fc receptor-positive cells have at least one Fcγ receptor I,II, or III.

[0017] The antibody used according to the invention is able to bind tomonocytes, macrophages, and/or dendritic cells being Fcγ receptorI-positive cells.

[0018] The antibodies used according to the invention lead to theinitiation or increase of the expression of CD40, CD80, CD86, ICAM-1,and/or LFA-3 being co-stimulatory antigens and/or secretion of cytokinsby the Fc receptor-positive cell. Preferably, the cytokins are IL-1,IL-2, IL-4, IL-6, IL-8, IL-12, and/or TNF-α.

[0019] Preferably, binding to the T cell takes place via the T cellreceptor complex of the T cell.

[0020] The bispecific antibody used in the invention preferably is ananti-CD3 X anti-tumour-associated antigen antibody and/or anti-CD2 Xanti-tumour-associated antigen antibody and/or anti-CD5 Xanti-tumour-associated antigen antibody and/or anti-CD28 Xanti-tumour-associated antigen antibody and/or anti-CD44 Xanti-tumour-associated antigen antibody.

[0021] The trispecific antibody used according to the inventionpreferably is an anti-CD3 X anti-tumour-associated antigen antibodyand/or anti-CD2 X anti-tumour-associated antigen antibody and/oranti-CD5 X anti-tumour-associated antigen antibody and/or anti-CD28 Xanti-tumour-associated antigen antibody and/or anti-CD44 Xanti-tumour-associated antigen antibody having an additional anti-Fcreceptor binding arm.

[0022] Regarding feature (a), the first signal is for example transducedvia the T cell receptor complex of the T cell and, therefore, may itselflead to an unphysiological T cell activation. By this, the cell isanergized and unable to react to T cell receptor-mediated stimuli. Inaddition, a second activation signal is transduced simultaneously to theT cell by the bispecific or trispecific antibodies of the invention viathe co-stimulatory antigens on the Fc receptor-positive cell whichcauses physiological activation of the T cell and, subsequently, leadsto killing of the tumour cell and/or proliferation of the T cell. As afurther criterion for T cell activation the up-regulation of cellsurface antigens such as CD2, CD25, and/or CD28, and/or the secretion ofcytokins such as IL-2 may be used.

[0023] Thus, by the use of the bsAb described according to the inventionT cells are activated and retargeted against the tumour cells.Generally, the use of an unspecific activation of T cells has been oflittle success in immune therapy.

[0024] Preferred antibodies are heterologous bispecific antibodiesselected of one or more of the following combinations of isotypes:

[0025] rat-IgG2b/mouse-IgG2a,

[0026] rat-IgG2b/mouse-IgG2b,

[0027] rat-IgG2b/mouse-IgG3;

[0028] rat-IgG2b/human-IgG1,

[0029] rat-IgG2b/human-IgG2,

[0030] rat-IgG2b/human-IgG3[oriental allotype G3m(st)=binding to proteinA],

[0031] rat-IgG2b/human-IgG4;

[0032] rat-IgG2b/rat-IgG2c;

[0033] mouse-IgG2a/human-IgG3[caucasian allotypes G3m(b+g)=no binding toprotein A, in the following indicated as *]

[0034] mouse-IgG2a/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0035]mouse-IgG2a/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0036] mouse-IgG2a/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0037] mouse-[VH-CH1, VL-CL]-human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0038] mouse-[VH-CH1, VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal region ofCH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3]

[0039] rat-IgG2b/mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge-CH2-CH3]

[0040] rat-IgG2b/mouse-[VH-CH1, VL-CL]-human-IgG2-[hinge-CH2-CH3]

[0041] rat-IgG2b/mouse-[VH-CH1, VL-CL]-human-IgG3-[hinge-CH2-CH3,oriental allotype]

[0042] rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG4-[hinge-CH2-CH3]

[0043] human-IgG1/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0044]human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3]

[0045] human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal region ofCH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3]

[0046] human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminal region ofCH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3]

[0047] human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminal region ofCH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3]

[0048] human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0049] human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0050] human-IgG2/human-[VH-CH1,VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-CH3]

[0051] human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH2-CH3]

[0052] human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal region ofCH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3]

[0053] mouse-IgG2b/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0054] mouse-IgG2b/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0055] mouse-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0056] The invention will be illustrated in more detail by means of theaccompanying Figures. The Figures show:

[0057]FIG. 1: The role of accessory cells in the immune therapy oftumours using bispecific antibodies;

[0058]FIG. 2: a diagram of the results obtained in the in vivaexperiment;

[0059]FIG. 3: an example of a bispecific antibody according to theinvention;

[0060]FIG. 4: trispecific F(ab)₂ antibody;

[0061]FIG. 5: trispecific scFv antibody.

[0062] The object of the invention aims to induction of an anti-tumourimmunity and in particular a long-lasting anti-tumour immunity byraising an efficient T cell response against tumour cells. This objecthas been achieved by redirection of T cells to tumour cells by means ofintact bispecific antibodies (bsAb) and concomitant binding of Fcreceptor-positive cells to the Fc portion of the bsAb. In this respect,it is important to note that the Fc receptor-positive cell is activatedby binding of immobilized intact bsAb (at the T cell or tumour cell,respectively) to the Fc receptor. “Long-lasting anti-tumour immunity”according to the invention is meant to be a period of time spanning atleast several years.

[0063] The bispecific and trispecifc antibodies described in the methodof the present invention are characterized in particular by the featuresdescribed in the Claims and, preferably, of course by the features(a)-(e) described in claim 1. Thus, these antibodies may be used for theinduction of an anti-tumour immunity, preferably a long-lastinganti-tumour immunity in humans and animals which will be particularlycaused by the features described under items (a)-(e) of claim 1.

[0064] Preferably, specific subclasses or combinations of subclasses,respectively, of the bsAb or in the case of trispecific antibodies abinding arm recognizing the Fc receptor are employed for the activationof the Fc receptor-positive cell by the bsAb. For example, in vitroexperiments showed that bsAb of the mouse-IgG2a/rat-IgG2b subclasscombination are able to bind to and simultaneously activate Fcreceptor-positive cells leading to up-regulation or new formation(expression), respectively, of co-stimulatory antigens such as CD40,CD80, or CD86 on the surface of these cells, whereas bsAb of themouse-IgG1/rat-IgG2b subclass combination are able to bind to Fcreceptor-positive cells (Haagen et al., Interaction of human monocyteFcγ receptors with rat IgG2b, J. Immunology, 1995, 154:1852-1860) butobviously are unable to activate these cells to a comparable extent(Gast G. C., Haagen I. -A., van Houten A. A., Klein S., Duits A. J., deWeger R. A., Vroom T. M., Clark M. R., J. Phillips, van Dijk A. J. G.,de Lau W. B. M., Bast B. J. E. G. CD8 T-cell activation afterintravenous administration of CD3×CD19 bispecific antibody in patientswith non-Hodgkin lymphoma. Cancer Immunol. Immunother. 40: 390, 1995).

[0065] While the intact bsAb binds to the T cell with one binding arm(e.g. CD3 or CD2) and activates it at the same time, co-stimulatorysignals from the Fc receptor-positive cell bound to the Fc portion ofthe bsAb can be transferred to the T cell. That means, only thecombination of T cell activation via one binding arm of the bsAb andsimultaneous mediation of co-stimulatory signals from the Fcreceptor-positive cell to the T cell leads to efficient T cellactivation (FIG. 1A).

[0066] Also, tumour-specific T cells which have been insufficientlyactivated at the tumour cell site and, therefore, remain in an anergicstate may be reactivated by the principle of the invention describedabove (FIG. 1B).

[0067] Thus, surprisingly by the inventive use of intact Tcell-redirecting bsAb a long-lasting anti-tumour immunity could beinduced according to the invention. This was based on the physiologicalactivation of tumour-retargeted T cells by 1) binding of the bsAb to theT cell, for example via the T cell receptor complex, and 2) simultaneoustransfer of co-stimulatory signals by FcR+ cells binding to the Fcportion of the bsAb.

[0068] Thus, an important prerequisite for an efficient induction ofanti-tumour immunity is the use of bsAbs having a Fc portion able tobind to FcR+cells which are activated themselves by this event and,thereby, able to mediate co-stimulatory signals to the T cell.

[0069] Due to this mechanism, tumour-specific anergic T cells (having aT cell receptor [TCR] which recognizes tumour-specific peptides inassociation with MHC molecules on the tumour cell) can be reactivated atthe tumour cell site, and thereby, the tumour tolerance can be reversed.

[0070] Since the tumour-specific T cells via their TCR are able torecognize completely different petides/proteins than the bsAb via itsanti-tumour binding arm, additional tumour cells may be recognized andkilled after activation of such T cells by the bsAb which have initiallynot been recognized by the bsAb. That means, it is not necessary for thebsAb used to recognize all of the tumour cells to induce an anti-tumourimmunity which afterwards includes all of the tumour cells. A furtherconclusion to be drawn from this knowledge is that a relatively lowamount of intact bsAb (100 μg-5 mg/patient) is sufficient to achieve theanti-tumour immunity of the invention. Since induction of such ananti-tumour immunity takes some time the patient should be in a minimalresidual disease (MRD) situation with a low amount of residual tumourcells at the start of therapy. MRD is intended to mean the period oftime after reduction of larger tumour portions using suitable methodssuch as surgical measures for removal of the primary tumour during whichresidual tumour cells still exist which possibly may not be detected butafter some time may lead to recurrence.

[0071] This has the following advantages for the type of therapydescribed herein:

[0072] The number of tumour cells and, thus, the possible appearance ofso-called “escape mutants”, i.e. for example of tumour variations nolonger presenting a tumour-specific peptide recognized by the immunesystem, is strongly reduced.

[0073] After “immunization” has taken place even very fast-growing andaggressive tumour variations are less apt to more or less overrun theimmune system.

[0074] The main differences compared to conventional cancer therapyusing bsAb are

[0075] 1. the lower amount of antibody administered (5 μg-10 mg,preferably 10 μg-100 μg, further 100 μg-5 mg/patient); and

[0076] 2. the time of administration of the bsAb with respect to thestage of the disease (preferably in a MRD situation). This type oftherapy does not wait until tumour cells are again detectable, forexample after removal of a primary tumour, but instead by theadministration of intact bsAb a kind of preventive immunization of thepatient against his own cancer cells is carried out;

[0077] 3. this is possible because with such low amounts of intact bsAbadministered no severe side effects have to be expected which wouldneutralize the enormous advantage—namely the absence of metastasesimpossible to cure;

[0078] 4. the induction of a humoral immune reaction accompanied bycomplement-fixing antibodies (isotypes IgG1, IgG2, and IgG3 in humans)able to kill tumour cells.

[0079] Preferably, the antibodies according to the invention aremonoclonal, chimeric, recombinant, synthetic, semi-synthetic, orchemically modified intact antibodies having for example Fv, Fab, scFv,or F(ab)₂ fragments.

[0080] If the antibodies used in the method of the present inventionshall be used in an in vivo therapy, preferably antibodies orderivatives or fragments of human origin are used, or antibodiesmodified to be suitable for the use in humans (so-called “humanizedantibodies”) (see for example Shalaby et al., J. Exp. Med. 175 (1992),217; Mocikat et al., Transplantation 57 (1994), 405).

[0081] The preparation of the different types of antibodies and antibodyfragments mentioned above is obvious to the skilled artisan. Thepreparation of monoclonal antibodies preferably of mammalian origin,e.g. of human, rat, mouse, rabbit, or goat, can be performed usingconventional methods for example as those described in Köhler undMilstein (Nature 256 (1975), 495), in Harlow und Lane (Antibodies, ALaboratory Manual (1988), Cold Spring Harbor) or in Galfré (Meth.Enzymol. 73 (1981), 3).

[0082] It is further possible to prepare the antibodies described bymeans of recombinant DNA technology according to techniques obvious tothe skilled artisan (see Kurucz et al., J. Immunol. 154 (1995), 4576;Hollinger et al., Proc. Natl. Acad. Sci. USA 90 (1993), 6444).

[0083] The preparation of antibodies having two different specificities,the so-called bispecific antibodies, can be performed for example usingrecombinant DNA technology but also by the so-called hybrid hybridomafusion technique (see for example Milstein et al., Nature 305 (1983),537). This technique consists of fusing hybridoma cell lines eachproducing antibodies having one of the desired specificities andidentifying and isolating recombinant cell lines producing antibodieshaving both specificities.

[0084] The problem forming the basis of the invention can be overcomeusing either bispecific or trispecific antibodies if they exhibit theproperties and effects characterized in claim 1. In the following, thepreparation of antibodies showing two and three specificities,respectively, is described in more detail. It is well-known from theprior art to provide such bispecific and trispecific antibodies, and theliterature describing such methods of preparation are incorporated herein their entirety by reference.

[0085] The preparation of antibodies exhibiting three specificities,so-called trispecific antibodies, also suitable to solve the basicproblem of the invention may for example be carried out by coupling athird antigen binding site having an additional specificity, e.g. in theform of “single chain variable fragments” (scFv) to one of the IgG heavychains of a bispecific antibody. The scFv may be coupled for exampleusing a

-S-S(G₄S)_(n)D-I-linker

[0086] to one of the heavy chains (S=serine, G=glycine, D=aspartate,I=isoleucine).

[0087] Analogously, trispecific F(ab)₂ constructs may be prepared byreplacing the CH2-CH3 regions of the heavy chain of one specificity of abispecific antibody by an scFv having a third specificity, while theCH2-CH3 regions of the heavy chain having the other specificity can beremoved for example by insertion of a stop codon (at the end of the“hinge” region) into the coding gene, e.g. by homologous recombination(see FIG. 4).

[0088] It is also possible to prepare trispecific scFv constructswherein three VH-VL regions representing three different specificitiesare arranged in series (FIG. 5).

[0089] According to the invention there are for example used intactbispecific antibodies. Intact bispecific antibodies are composed of twoantibody semi-molecules (each having a H and a L immunoglobulin chain)each representing a specificity, and additionally like normal antibodieshaving a Fc portion performing the well-known effector functions. Theyare preferably prepared using the quadroma technology. This method ofpreparation is exemplified in DE-A-44 19 399. For complete disclosurethis document is incorporated in its entirety by reference also withrespect to a definition of bispecific antibodies. It should beunderstood that other methods of preparation are also useful if theylead to the intact bispecific antibodies according to the abovedefinition required according to the invention.

[0090] For example, intact bispecific antibodies may be produced insufficient amounts using a newly developed method of preparation (1).The combination of two bispecific antibodies directed against twodifferent tumour-associated antigens (e.g. c-erb-B2, ep-cam, such asGA-733-2=C215) on the mamma carcinoma cells minimizes the risk thattumour cells expressing only one of the antigens remain unidentified.

[0091] Bispecific antibodies are able to bind to the T cell receptorcomplex on the T cell with one binding arm and to tumour-associatedantigens on the tumour cell with the second binding arm. Thereby, Tcells are activated which kill the tumour cells by releasing cytokins orby apoptosis-mediating mechanisms. In addition, there is the possibilitythat T cells recognize tumour-specific antigens by their receptor duringactivation by the bispecific antibodies and, thereby, a long-lastingimmunisation is initiated (FIG. 1B). Of particular importance in thisrespect is the intact Fc portion of the bispecific antibody whichmediates the binding to accessory cells such asmonocytes/makrophages/dendritic cells and causes these cells to developcytotoxicity, and/or concomitantly transfer important co-stimulatorysignals to the T cell (FIG. 1).

[0092] In this manner, a T cell reaction may also be induced againsttumour-specific peptides which are unknown to date.

[0093] By redirection of possibly anergized tumour-specific T cells totumour cells by the bsAb and concurrent co-stimulation of such cells byaccessory cells binding to the Fc portion of the bsAb the anergic stateof the CTLs may be reversed. I.e. according to the invention apreexisting T cell tolerance in the patient against the tumour isreversed by means of intact bsAb and, thus, a long-lasting anti-tumourimmunity is induced.

[0094] This is supported by initial in vivo data from experiments withmice pointing to such a long-lasting anti-tumour immunity aftertreatment with a syngeneic tumour and intact bsAb. In these experiments,14 out of 14 test animals which were successfully treated with bsAbfollowing a first tumour injection survived a further tumour injectioncarried out 144 days after the first injection—without additionaladministration of bsAb (see Example 1).

[0095] The immunisation strategy according to the invention uses forexample intact bsAb able to bind to FcγTRI+ cells via their Fc portion.In addition, the bsAb used according to the invention have as a secondspecificity besides the tumour cell-binding specificity for exampleanti-CD3, i.e. they are able to bind to T cells via the second bindingarm. Thus, by the bsAb used according to the invention T cells areactivated and redirected against the tumour cells.

[0096] It has been attempted to achieve anti-tumour immunity bytreatment with bispecific F(ab′)2 fragments having the specificitiesanti-c-erb-B2×anti-CD64. The main disadvantage of bsF(ab′)2 fragments isthat due to the specificities used merely FcγTRI+ cells are redirectedto the tumour. T cells are not redirected to the tumour by thisbispecific antibody. The bsF(ab′)2 fragments have the potential todirectly kill the tumour but themselves are not able to establishanti-tumour immunity. Only the T cell with its specific T cell receptoris able to confer anti-tumour immunity. The FcγTRI+ cells are able toindirectly activate tumour-specific T cells by presentation oftumour-specific peptides (via MHC I or MHC II, respectively) for exampleafter phagocytosis of tumour cell components but the efficiency ofinduction of anti-tumour immunity in this case is not as high (only in30% of patients).

[0097] Further advantages of intact bsAb able to redirect T cellscompared to the above-mentioned bsF(ab′)2 fragments are detailed asfollows:

[0098] 1. It is possible for Fc receptor-positive cells to bind tointact bsAb and on the one hand to contribute directly to tumour killingvia ADCC (antibody-dependent cell-mediated cytotoxicity) and on theother hand contribute to T cell activation as detailed above.

[0099] 2. The circulation time of bsAb with Fc portion is significantlylonger than for example of bsF(ab′)2 or bs(scFv) antibody fragments sothat considerably lower doses of intact ab molecules are required toachieve a comparable anti-tumour effect.

[0100] 3. The use according to the invention of intact bispecificantibodies is in particular directed to the killing of residual tumourcells/mikrometastases. In contrast to the treatment of solid tumours orlarger metastases in which the above-mentioned antibody fragments may beadvantageous since they allow for better penetration of the tumour dueto their smaller size.

[0101] 4. Intact T cell-redirecting bsAb function also in directinganergized tumour-specific T cells to the tumour cell which according tothe invention may be reactivated directly at the tumour site. This isimpossible to achieve using a bsF(ab′)2 fragment having thespecificities anti-CD64 X anti-tumour-associated antigen.

[0102] 5. A bsF(ab′)2 fragment having the specificities anti-CD64 Xanti-tumour-associated antigen is only able to achieve anti-tumourimmunity in 30% of the patients while according to the invention inexperiments with mice a protection of 100% of the animals could beachieved using T cell-redirecting intact bsAb.

[0103] The binding of bsAb to Fcγ-RI shows two essential advantages withregard to an optimal anti-tumour effectivity: Fcγ-RI-positive cells havethe ability to eliminate tumour cells by ADCC (2) and, thus, are able tocontribute synergistically to the anti-tumour effect of the cytotoxic Tcells directed to the tumour by the bsAb (3).

[0104] 1. FcγRI-positive cells (such as monocytes/macrophages/dendriticcells) are able to provide important co-stimulatory signals similar toantigen presentation to the T cell and, thereby, prevent anergizing ofthe T cell. Furthermore, as shown in FIG. 1, even T cells having a Tcell receptor which recognizes tumour-specific peptides (presented viaMHC antigens on the tumour cell) can be stimulated as a desiredby-product due to the bsAb-mediated interaction of the T cell withaccessory cell and tumour cell. In this constellation, the co-stimulinecessary for correct activation of the T cell would be provided by theaccessory cell (such as the monocyte). Thus, besides the direct T cellreceptor-independent bsAb-mediated tumour killing (FIG. 1A) the solutionof the invention should also be able to activate and generatetumour-specific T cells (FIG. 1B) which after degradation of the bsAbcontinue to patrol in the patient. That means, similar togenetherapeutic approaches (e.g. by incorporation of co-stimulatoryantigens such as B-7 in the tumour cell) by intact bsAb the tumourtolerance in the patient can be reversed.

[0105] In this respect it is further advantageous that the expression ofFcγ-RI on the respective cells is up-regulated after G-CSF treatment.

EXAMPLE

[0106] To investigate the question of induction of an anti-tumourimmunity by bispecific antibodies C57BL/6 mice were first injected with5×10³ syngeneic B16 tumour cells. Two days later, one group of mice(n=18) was treated with an intact bsAb prepared using quadromatechnology (1) and which recognizes an epCAM target structure(C215=tumour-associated antigen) on the tumour cell as well as CD3 on Tcells. This bsAb was of the IgG2a/rat IgG2b subclass combination. Asecond group (n=6) only received an equimolar amount of Fab fragments ofthe two specificities contained in the bsAb. While all of the animals ofthe Fab control group died within 56 days or had to be put to sleep, 14out of the 18 animals treated with bsAb survived. The surviving animalsreceived an i.p. injection of an additional dose of 750 B16 tumour cells144 days after the first injection of tumour cells but this time withoutadministration of bsAb. The same amount of tumour cells was administeredto five untreated animals serving as a control. While the last animal ofthe untreated control group had to be put to sleep 66 days after tumourinjection, all of the animals treated with bsAb survived (monitoringperiod: 120 days after second injection of tumour cells). See also FIGS.2A and 2B: Survival curves of the two successive experiments describedabove.

[0107] These experiments show that it is possible to induce along-lasting anti-tumour immunity using the bispecific antibodies andalso the trispecific antibodies provided according to the invention. Itcan be expected that these events may also be applied to humans in whichcase a long-term anti-tumour immunity lasting at least several years canbe expected.

Detection of the Induction of a Humoral Immune Reaction by Intact bsAband the Importance Thereof for the Prognosis of the Survival of Patientsor Animals Description

[0108] Besides the cellular immunity mainly kept up by the T cells thehumoral immunity plays a similar important role in tumour recognitionand killing. Evidence for this comes from a number of investigations,for example the work of Rodolfo et al. (J. Immunol. 157, 5536, 1996).Rodolfo et al. were able to show that (1) tumour-specific T cells areinduced by administration of tumour cell vaccine transduced by IL-12 orIL-2, respectively; (2) an effective humoral immune reaction withcomplement-fixing antibodies could only be generated using IL-12transduced tumour cells. Interestingly, in these experiments only theanimals survived which had developed complement-fixing antibodies withanti-tumour specificity.

[0109] By the in vitro experiments carried out according to theinvention we were able to show that using the intact bispecificantibodies described herein due to their binding and activation of Fcγreceptor-positive cells (see FIG. 1) the latter were able to generateand secrete cytokins such as IL-12 (see Annex Table 1). Thus, by theintact bsAb described herein it was possible to induce the production ofcytokins without gene transfer which otherwise can only be expressedusing gene transfer in the framework of genetherapeutic approaches. Thisobservation is of importance because IL-12 obviously plays an importantrole in the induction of an anti-tumour immunity.

[0110] In the animal experiments shown in the Example described abovetumour-reactive complement-fixing antibodies could be detected in tailblood of the surviving mice after bsAb treatment on day 143 after tumouradministration. This shows that such antibodies at least contributed tothe survival of the animals since in the deceased animals no or only lowamounts of these antibodies could be detected (Table 2).

[0111] The experiments described above were performed using bispecificantibodies. Instead of the bispecific antibodies, it is also possible touse the trispecific antibodies described in the specification to obtainsimilar results.

[0112] As described, by treatment with the bispecific and trispecificantibodies provided according to the invention tumour-reactivecomplement-binding antibodies are generated being indicative for theinduction of a humoral immune reaction. This together with the inductionof the production of cytokins such as IL-12 probably at leastcontributes to the induction of long-term anti-tumour immunity accordingto the invention. Therefore, it is also possible to use the amount ofcomplement-binding tumour-reactive antibodies induced by the treatmentwith bispecific or trispecific antibodies according to the invention forthe assessment of the course of the disease (prognosis) of a tumourpatient. The higher the amount of tumour-reactive complement-fixingantibodies, the more favourable is the prognosis of the tumour patient.

[0113] The use of for example intact bispecific antibodies disclosedaccording to the invention is carried out after treatment of a primarytumour, preferably in patients being in a minimal residual disease (MRD)situation. The use of intact bispecific antibodies according to theinvention will only gain importance in patients having a low amount ofresidual tumour cells but with a high risk of recurrence.

[0114] The term “tumour immunity” comprises the induction for a humuraland/or cellular immunity directed against the tumour of a human oranimal suffering from tumour disease. This means that tumour specificantibodies and/or tumour specific T cells or other immune cells areinduced which are directed against the tumour. TABLE 1 Increase insurface antigens on CD14+ cells (makrophages, monocytes) or secretion ofcytokins, respectively, after in vitro incubation of PMBC with intactbsAb and tumour cells after 20 h. Antigen without ab with bsAbCD40 + + + CD80 (B-7.1) + + + CD86 (B-7.2) + + + CD25 (IL-2R) + + + +MHC II + + (+) bsAb − +

[0115] TABLE 2 B16 melanoma rechallege experiment (see Example 1). Testof sera (from tail blood) of surviving animals (1-13) and the tumours ofdeceased animals (14-17) for tumour-reactive antibodies. MouseReactivity Reactivity Reactivity Comple- non- with with with ment-Comple- B16-C215 B16-C215 B16-C215 fixing ment- cells % cells % cells %subclass fixing (serum of (serum of (serum of mIgG2a subclass day 0) dayof death day 143) mIgG1 or putting to sleep, respectively)

Control Serum

[0116] a) The measurements were carried out in a FACScan(Becton-Dickinson). The tumour-bound antibodies were detected usingpolyclonal fluorescence-labeled rat anti-mouse antibodies. The numericalvalue is a measure for fluorescence intensity

[0117] b) Investigation of murine tumour-reactive sera as under a) butusing isotype-specific detecting antibodies directed against mIgG2a(complement-fixing) or mIgG1(non-complement-fixing), respectively

References

[0118] 1. Lindhofer et al, Preferential species-restricted heavy-lightchain pairing in rat-mouse quadromas: Implications for a single steppurification of bispecific antibodies, J. Immunology 1995, 155:219

[0119] 2. Valerius et al., Involvement of the high-affinity receptor forIgG (FcγRI; CD64) in enhanced tumor cell cytotoxicity of neutrophilsduring granulocyte colony-stimulating factor therapy, Blood, 1993,82:931-939

[0120] 3. Weiner et al.,The role of T cell activation in anti-CD3 Xantitumor bispecific antibody therapy, J. Immunology, 1994, 152:2385

1. A method for the induction of an anti-tumor immunity by administeringto a human or animal an efficient amount of an intact bispecific ortrispecific antibody having the following properties and effects of: (a)binding to a T cell and mediating a first activation signal thereto; (b)binding to tumour-specific antigens on a tumour cell; (c) binding,through its Fc portion (in the case of bispecific antibodies) or a thirdspecificity (in the case of trispecific antibodies) to the Fc receptorof Fc receptor-positive cells; (d) activation of the Fcreceptor-positive cell by binding to the Fc receptor-positive cell and,thereby initiating or increasing the expression of cytokins and/orco-stimulatory antigens; (e) transfer of at least a second activationsignal required for pysiological activation of the T cell to the T cellby the co-stimulatory antigens and/or cytokins, this activation beingindicated by up-regulation of activation markers, killing of the tumourcell, and/or T cell proliferation.
 2. Method according to claim 1,characterized in that said antibody further is able to reactivatetumour-specific T calls being in a state of anergy.
 3. Method accordingto claim 1 or 2, characterized in that said antibody further is able toactivate the tumour-specific T cells recognizing a tumour-specificpeptide presented on the tumour cells by MHC class I and/or class II viatheir T cell receptor upon binding to the bispecific or trispecificantibody as described under (e).
 4. Method according to claim 1, 2 or 3,characterized in that said antibody is able to induce tumour-reactivecomplement-binding antibodies and, thereby, to induce a humoral immunereaction.
 5. Method according to claim 1, 2 or 3, characterized in thatsaid antibody binds to the T cell via CD3 or CD2, CD5, CD28, and/orCD44.
 6. Method according to claim 1, 2 or 3, characterized in that saidantibody is able to bind to Fc receptor-positive cells having a Fcγreceptor I, II, or III.
 7. Method according to claim 6, characterized inthat said antibody is able to bind to monocytes, makrophages and/ordendritic cells being Fcγ receptor I-positive cells.
 8. Method accordingto claim 1, 2 or 3, characterized in that by the Fc receptor-positivecell the expression of the co-stimulatory antigens CD40, CD80, CD86,ICAM-1, and/or LFA-3 and/or the secretion of cytokins is initiated orincreased.
 9. Method according to claim 8, characterized in that thecytokins are IL-1, IL-2, IL-4, IL-6, IL-8, IL-12, and/or TNF-α. 10.Method according to claim 1, characterized in that said binding to the Tcell takes place via the T cell receptor complex of the T cell. 11.Method according to claim 1, characterized in that said bispecificantibody is an anti-CD3 X anti-tumour-associated antigen antibody and/oranti-CD2 X anti-tumour-associated antigen antibody and/or anti-CD5 Xanti-tumour-associated antigen antibody and/or anti-CD28 X anti-tumour-associated antigen antibody and/or anti-CD44 X anti-tumour -associatedantigen antibody.
 12. Method according to claim 1, characterized in thatsaid bispecific antibody is a heterologous rat/mouse bispecificantibody.
 13. Method according to claim 1, characterized in that saidbispecific antibody is a heterologous bispecific antibody selected ofone or more of the following combinations of isotypes:rat-IgG2b/mouse-IgG2a, rat-IgG2b/mouse-IgG2b, rat-IgG2b/mouse-IgG3,rat-IgG2b/human-IgG1, rat-IgG2b/human-IgG2,rat-IgG2b/human-IgG3[oriental allotype G3m(st)=binding to protein A],rat-IgG2b/human-IgG4, rat-IgG2b/rat-IgG2c,mouse-IgG2a/human-IgG3[caucasian allotypes G3m(b+g)=no binding toprotein A, in the following indicated as *] mouse-IgG2a/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3] mouse-IgG2a/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]mouse-IgG2a/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3] mouse-[VH-CH1,VL-CL]-human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3] mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal region ofCH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3] rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge-CH2-CH3] rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG2-[hinge-CH2-CH3] rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG3-[hinge-CH2-CH3, oriental allotype]rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG4-[hinge-CH2-CH3]human-IgG1/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3] human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal region ofCH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3] human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal region ofCH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3]human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3] human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminal region ofCH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3] human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]human-IgG2/human-[VH-CH1,VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-CH3]human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH2-CH3]human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2:>aa position251]-human-IgG3*[CH3] mouse-IgG2b/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]mouse-IgG2b/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]mouse-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3].
 14. Method according toclaim 1, characterized in that said trispecific antibody is an anti-CD3X anti-tumour-associated antigen antibody and/or anti-CD2 Xanti-tumour-associated antigen antibody and/or anti-CD5 Xanti-tumour-associated antigen antibody and/or anti-CD28 Xanti-tumour-associated antigen antibody and/or anti-CD44 Xanti-tumour-associated antigen antibody having an additional anti-Fcbinding arm.
 15. Method according to claim 1 for the prophylaxis andtreatment of tumour diseases.
 16. Method according to claim 1 whereinsaid antibody is administered in an amount of 5 μg-10 mg/patient byapplication to the patient being in a minimal residual disease (MRD)situation.